Benzotriazines, pharmaceutical compositions, and methods of using same

ABSTRACT

Use of compounds of the formula I ##STR1## in which R 1  signifies an amino, C 1  -C 6  -alkylamino, di-C 1  -C 6  -alkylamino or a hydroxyl group, R 2  and R 3 , which are the same or different, each hydrogen, halogen, C 1  -C 6  -alkyl, C 1  -C 6  -alkoxy, cyano, carboxyl, halogen-C 1  -C 6  -alkyl, cyano-C 1  -C 6  -alkyl, carboxy-C 1  -C 6  -alkyl, C 1  -C 6  - alkyl, hydroxyl, carboxy-C 1  -C 6  -alkoxy, C 1  -C 6  -alkoxycarbonyl-C 1  -C 6  -alkoxy or aminocarbonyl-C 1  -C 6  -alkoxy, C 1  -C 6  -alkylaminocarbonyl-C 1  -C 6  -alkoxy or di-C 1  -C 6  -alkylaminocarbonyl-C 1  -C 6  -alkoxy and n the number 0 or 1, their tautomers, as well as salts with non-toxic acids or bases, for the preparation of medicaments with PLA 2  -inhibiting action, new compounds and process for their preparation.

This is a National Stage Entry Application, filed under 35 U.S.C. 371,of PCT/EP93/03542, filed Dec. 15, 1993.

The subject of the present invention is the use of benzotriazines asPLA₂ inhibitors, new benzotriazine derivatives, process for theirpreparation and medicaments which contain these compounds.

The invention concerns the use of benzotriazine derivatives of thegeneral formula I ##STR2## for the preparation of medicaments with PLA₂-inhibiting action, whereby R₁ signifies an amino, C₁ -C₆ -alkylamino,di-C₁ -C₆ -alkylamino or a hydroxyl group, R₂ and R₂, which are the sameor different, each hydrogen, halogen, C₁ -C₆ -alkyl, C₁ -C₆ -alkoxy,cyano, carboxyl, halogen-C₁ -C₆ -alkyl, cyano-C₁ -C₆ -alkyl, carboxy-C₁-C₆ -alkyl, hydroxyl, carboxy-C₁ -C₆ -alkoxy, C₁ -C₆ -alkoxycarbonyl-C₁-C₆ -alkoxy or aminocarbonyl-C₁ -C₆ -alkoxy, C₁ -C₆-alkylaminocarbonyl-C₁ -C₆ -alkoxy or di-C₁ -C₆ -alkylamino-carbonyl-C₁-C₆ -alkoxy and n the number. 0 or 1, their tautomers, as well as saltswith non-toxic acids or bases.

Compounds of the formula I are in part known. Thus, e.g. in J. Amer.Chem. Soc. 76, 3551 (1954), compounds of the formula I with R₂ =Cl or Hand R₁ =OH or NH₂, R₃ =H and n=O are described as anti-malarial agents.Further compounds with n=1 and R₁ =NH₂ are known from J. Chem. Soc. (B)1970. 911. In these documents, they are described as dyestuffs,radiosensitisers, bactericides, insecticides, acaricides, cytotoxicagents and anti-malarial agents. An anti-inflammatory action is notstated.

The compounds of the formula I display valuable pharmacologicalproperties, especially they can inhibit the activity of phospholipases.Therefore, they are suitable for the treatment of acute and chronic,allergic, non-allergic and traumatic inflammatory diseases, such as forexample rheumatic arthritis, osteoarthritis, ulcerative colitis, acutepancreatitis, contact dermatitis, inflammatory and allergic respirarorydiseases, septic shock, allergic shock, serum disease, auto-immunediseases, graft-versus-host reactions, host-versus-graft diseases,ischaemic or thrombotic diseases, for example coronary infarct orcerebral infarct.

The "alkyl parts" in the aliphatic groups mentioned in the case of R²and R³ can be straight-chained or branched. Preferred radicals are themethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert.-butyl,n-pentyl and 3-pentyl radical. As C₁ -C₆ -alkoxy group, in this sensethere come into question, for example, the methoxy, ethoxy, propoxy orbutoxy group.

Halogen atoms are especially fluorine, chlorine and bromine.

Apart from the compounds mentioned in the Examples, the subject of theinvention are especially all substances which display all possiblecombinations of the substituents mentioned in the Examples.

The subject of the invention are also new compounds of the formula Ia##STR3## in which R₂ signifies C₁ -C₆ -alkyl, C₁ -C₆ -alkoxy, cyano,halogen-C₁ -C₆ -alkyl, cyano-C₁ -C₆ -alkyl, carboxy-C₁ -C₆ -alkyl,hydroxyl, carboxy-C₁ -C₆ -alkoxy, C₁ -C₆ -alkoxy-carbonyl-C₁ -C₆-alkoxy, aminocarbonyl-C₁ -C₆ -alkoxy, C₁ -C₆ -alkylaminocarbonyl-C₁ -C₆-alkoxy or di-C₁ -C₆ -alkylaminocanbonyl-C₁ -C₆ -alkoxy and R₃ hydrogenor C₁ -C₆ -alkyl, their tautomers, as well as salts with non-toxic acidsor bases.

The compounds of the formula I and Ia, respectively, are especiallysuitable for the preparation of medicaments for the treatment of acuteor chronic diseases, especially those diseases with inflammatory,immunological, allergic, non-allergic or traumatic genesis. Sincephospholipase A₂ (PLA₂) is a key enzyme in the causation of these:diseases, such diseases are effecively treated by inhibition of thisenzyme. Surprisingly, it has now been found that the compounds of theformula I or Ia, respectively, inhibit phospholipase A₂ and thus, on thebasis of this new action profile, can be used in the treatment ofdiseases in which the inhibition of phospholipase A₂ is of clinical,pathological or symptomatic relevance., especially in the case ofinflammatory diseases.

In the case of compounds of the formula I, R¹ preferably signifies anamino or hydroxyl group.

In the case of the compounds of the formula I or Ia, the substituents R²and R³ can, independently of one another, stand in the 5-, 6-, 7- or8-position of the benztriazole system. Preferably, there come intoquestion derivatives monosubstituted on the phenyl ring (R³ =hydrogenand R² not hydrogen). In the case of disubstituted derivatives (neitherR² nor R³ hydrogen), the substituents R² and R³ preferably stand in the6- and 7-position of the benztriazole system.

Preferred radicals for R² are especially the following substituents:chlorine, methyl, ethyl, isopropyl, t-butyl, methoxy,1,1-dimethylcyanomethyl, hydroxyl, carboxymethoxy. R³ preferablysignifies hydrogen or the methyl group.

The process according to the invention for the preparation of thecompounds of the formula I or Ia, respectively, is characterised inthat, in per se known way, one reacts a compound of the general formulaII ##STR4## in which R₂ and R₃ have the above-mentioned meaning, withcyanamide and cyclises under basic conditions and subsequently convertsthe product obtained by reduction and subsequent diazotisation andhydrolysis into a compound of the formula I, in which R₂ and R₃ have theabove-mentioned meaning, as well as possibly converts into a salt byneutralisation with non-toxic acids or bases.

The reaction of compounds of the formula II with cyanamide expedientlytakes place in aqueous medium with proton catalysis and warming, forexample in conc. hydrochloric acid, the subsequent cyclisation in abasic medium with warming, for example in semiconc. caustic soda lye.

A reduction if desired to be carried out takes place expediently in asolvent, such as for example a lower alcohol or water or a mixturethereof, such as for example dil. ethanol, with a reducing agent, suchas sodium dithionite. However, it can also be carried out in a solvent,such as a lower alcohol, with catalytically activated hydrogen withnickel catalysis or in acetic acid with iron powder or zinc.

A diazotisation if desired to be carried out expediently takes placewith nitrous acid, prepared from alkali metal nitrites, such as e.g.sodium nitrite, in acids, such as e.g. acetic acid, hydrochloric acid orsulphuric acid, at temperatures between -20° C. and +30° C. For thehydrolysis, it can be left to stand in aqueous medium or, if desired,warmed.

As pharmacologically compatible salts , there come into questionespecially alkali metal, alkaline earth metal and ammonium salts, aswell as possibly salts with non-toxic inorganic or organic salts, suchas e.g. hydrochloric acid, sulphuric acid, phosphoric acid, hydrobromicacid, acetic acid, lactic acid, citric acid, malic acid, benzoic acid,salicylic acid, malonic acid, maleic acid, succinic acid ordiaminocaproic acid.

One obtains the salts in the usual way, e.g. by neutralisation of thecompounds of the formula I with the corresponding lyes or acids.

For the preparation of medicaments, the compounds of the general formulaI are mixed in per se known manner with suitable pharmaceutical carriersubstances, aroma, flavouring and colouring materials and formed, forexample as tablets or dragees, or, with addition of appropriateadjuvants, suspended or dissolved in water or oil, such as e.g. oliveoil.

The substances of the general formula I or Ia can be administered orallyand parenterally in liquid or solid form. As injection medium, there ispreferably used water which contains the stabilising agents,solubilising agents and/or buffers usual in the case of injectionsolutions. Such additives are e.g. tartrate or borate buffers, ethanol,dimethyl sulphoxide, complex formers (such asethylenediamine-tetraacetic acid), high molecular polymers (such asliquid polyethylene oxide) for viscosity regulation or polyethylenederivatives of sorbitol anhydrides.

Solid carrier materials are e.g. starch, lactose, mannitol, methylcellulose, talc, highly dispersed silicic acid, high molecular polymers(such as polyethylene glycols).

Compositions suitable for oral administration can, if desired, containedflavouring and sweetening materials. For the external use, thesubstances I according to the invention can also be used in the form ofpowders and salves. For this purpose, they are mixed e.g. with powdered,physiologically compatible dilution agents or usual salve bases. Theadministered dose depends upon the age, the state of health and theWeight of the recipient, the extent of the disease, the nature offurther treatments possibly carried out simultaneously, the frequency ofthe treatments and the nature of the desired action. Usually, the dailydose of the active compound amounts to 0.1 to 50 mg/kg of body weight.Normally, 0.5 to 4.0 and preferably 1.0 to 20 mg/kg/day in one or moreapplications per day are effective in order to obtain the desiredresults.

Apart from the substances mentioned in the Examples, the followingcompounds are preferred in the meaning of the present invention:

7-isoproipyl-12,4-benzotriazin-3-ol

7-t-butyl-1,2,4-benzotriazin-3-ol

6,7-dimethyl-1,2,4-benzotriazin-3-ol

7-hydroxy-1,2,4-benzotriazin-3-ol

6-chloro-1,2,4-benzotriazin-3-ol

7-chloro-1,2,4-benzotriazin-3-ol

7-methoxy-1,2,4-benzotriazin-3-ol

7-carboxymethoxy-1,2,4-benzotriazin-3-ol

EXAMPLE 1 1,2,4-Benzotriazine-3-amine 1-oxide

A mixture of 10 g 2-nitroaniline and 20 g cyanamide is heated for 5 minto 100° C., cooled, mixed with 25 ml conc. hydrochloric acid andcarefully heated to 70° C. After subsidence of the vigorous reaction,one cools to room temp., adds dropwise thereto 50 ml semiconce. causticsoda lye, heats for 30 min to 100° C., cools, filters and washes theprecipitate with hot glacial acetic acid. There remain 9.1 g of titlecompound (78% of theory) of the m.p. 272°-274° C.

EXAMPLE 2

In a manner analogous to that described in Example 1, one obtains:

    ______________________________________                        yield  melting point °C.    designation         (%)    (solvent)    ______________________________________    a)  6-chloro-1,2,4-benzotriazine-                            58     above 300        3-amine 1-oxide from 5-chloro-                                   (glacial acetic        2-nitroaniline             acid)    b)  7-chloro-1,2,4-benzotriazine-                            81     above 280        3-amine 1-oxide from 4-chloro-                                   (glacial acetic        2-nitroaniline             acid)    c)  5-methyl-1,2,4-benzotriazine-                            72     266-268        3-amine 1-oxide from 3-methyl-                                   (glacial acetic        6-nitroaniline             acid)    d)  6-methyl-1,2,4-benzotriazine-                            77     284-286        3-amine 1-oxide from 5-methyl-                                   (glacial acetic        2-nitroaniline             acid)    e)  7-methyl-1,2,4-benzotriazine-                            98     276-278        3-amine 1-oxide from 4-methyl-                                   (glacial acetic        2-nitroaniline             acid)    f)  8-methyl-1,2,4-benzotriazine-                            96     277-279        3-amine 1-oxide from 3-methyl-                                   (glacial acetic        2-nitroaniline             acid)    g)  7-ethyl-1,2,4-benzotriazine-                            94     225-227        3-amine 1-oxide from 4-ethyl-                                   (glacial acetic        2-nitroaniline             acid)    h)  7-isopropyl-1,2,4-benzo-                            87     250-252        triazine-3-amine 1-oxide   (glacial acetic        from 4-isopropyl-2-nitro-  acid)        aniline    i)  7-t-butyl-1,2,4-benzotriazine-                            77     143-145        3-amine-1-oxide from 4-t-  (ethyl        butyl-2-nitroaniline       acetate)    j)  6,7-dimethyl-1,2,4-benzo-                            97     above 270        triazine-3-amine 1-oxide   (glacial acetic        from 4,5-dimethyl-2-       acid)        nitroaniline    k)  5-methoxy-1,2,4-benzo-                            91     above 270        triazine-3-amine 1-oxide   (glacial acetic        from 2-methoxy-6-nitro-    acid)        aniline    l)  7-methoxy-1,2,4-benzo-                            96     273-274        triazine-3-amine 1-oxide   (glacial acetic        4-methoxy-2-nitroaniline   acid)    m)  7-(1,1-dimethylcyano-                            98     226-228        methyl)-1,2,4-benzo-       (water)        triazine 3-amine 1-oxide        from 4-(1,1-dimethylcyano-        methyl)-2-nitroaniline    n)  7-hydroxy-1,2,4-benzo-                            95     above 270        triazine-3-amine 1-oxide   (glacial acetic        from 4-hydroxy-2-nitro-    acid)        aniline    ______________________________________

EXAMPLE 3 1,2,4-Benzotriazine-3-amine

A mixture of 8.1 g (49 mmol) 1,2,4-benzotriazine-3-amine 1-oxide, 8.1 gsodium dithionite and 800 ml 70 percent ethanol is heated under refluxfor 1 h. One filters hot, evaporates the filtrate, washes the residuewith water and dries. There remain 7.0 g 1,2,4-benzotriazine-3-amine(98% of theory) of the m.p. 208°-210° C.

EXAMPLE 4

In a manner analogous to that described in Example 3, one obtains:

    ______________________________________                        yield  melting point °C.    designation         (%)    (solvent)    ______________________________________    a)  5-methyl-1,2,4-benzotriazine-                            97     198-200        3-amine from compound of   (water)        Example 2c    b)  6-methyl-1,2,4-benzotriazine-                            95     238-240        3-amine from compound of   (water)        Example 2d    c)  7-methyl-1,2,4-benzotriazine                            100    212-214        3-amine from compound of   (water)        Example 2e    d)  8-methyl-1,2,4-benzotriazine-                            86     260-262        3-amine from compound of   (water)        Example 2f    e)  7-ethyl-1,2,4-benzotriazine-                            59     164-166        3-amine from compound of   (water)        Example 2g    f)  5-methoxy-1,2,4-benzotriazine                            94     230-232        3-amine from compound of   (water)        Example 2k    ______________________________________

EXAMPLE 5 1,2,4-Benzotriazin-3-ol

To a solution of 2.0 g (14 mmol) 1,2,4-benzotriazine-3-amine in 30 mlwater and 3 ml conc. sulphuric acid one adds dropwise at 10° C. asolution of 1 g sodium nitrite in 10 ml water. One stirs for 4 b at roomtemp., filters off the residue and dries. One obtains 1.1 g of titlecompound (55% of theory) of the m.p. 204-206° C.

EXAMPLE 6

In a manner analogous to that described in Example 5, one obtains:

    ______________________________________                       yield  melting point °C.    designation        (%)    (solvent)    ______________________________________    a)  5-methyl-1,2,4-benzotriazin-                           95     206-208        3-ol from compound of     (water)        Example 4a    b)  6-methyl-1,2,4-benzotriazin-                           73     above 300        3-ol from compound of     (ether)        Example 4b    c)  7-methyl-1,2,4-benzotriazin-                           54     202-204)        3-ol from compound of     (ether)        Example 4c    d)  8-methyl-1,2,4-benzotriazin-                           62     195-197        3-ol from compound of     (ether)        Example 4d    e)  7-ethyl-1,2,4-benzotriazin-                           39     180-182        3-ol from compound of     (isohexane)        Example 4e    f)  5-methoxy-1,2,4-benzotriazin-                           57     186-188        3-ol from compound of     (ether)        Example 4f    ______________________________________

EXAMPLE 7 1,2,4-Benzotriazin-3-ol 1-oxide

To a solution of 7.5 g (46 mmol) 1,2,4-benzotriazine-3-amine 1-oxide(Chem. Ber. 46, 3522 (1913) in 75 ml water and 27 ml conc. sulphuricacid one adds dropwise at room temp. a solution of 13.3 g sodium nitritein 25 ml water. One leaves to stand overnight, filters, takes up in sodasolution, washes with ether, acidifies the aqueous phase, filters andrecrystallises from glacial acetic acid. One isolates 4.7 g of titlecompound (63% of theory) of the m.p. 238-239° C.

EXAMPLE 8

In a manner analogous to that described in Example 7, one obtains:

    ______________________________________                        yield  melting point °C.    designation         (%)    (solvent)    ______________________________________    a)  6-chloro-1,2,4-benzotriazin-                            87     209-211        3-ol 1-oxide from compound (water)        of Example 2a    b)  7-chloro-1,2,4-benzotriazin-                            62     241-243        3-ol 1-oxide from compound (water)        of Example 2b    c)  5-methyl-1,2,4-benzotriazin-                            51     235-237        3-ol 1-oxide from compound (water)        of Example 2c    d)  6-methyl-1,2,4-benzotriazin-                            77     238-240        3-ol 1-oxide from compound (water)        of Example 2d    e)  7-methyl-1,2,4-benzotriazin-                            64     242-244        3-ol 1-oxide from compound (water)        of Example 2e    f)  8-methyl-1,2,4-benzotriazin-                            65     236-238        3-ol 1-oxide from compound (water)        of Example 2f    g)  7-ethyl-1,2,4-benzotriazin-                            33     184-186        3-ol 1-oxide from compound (isopropanol)        of Example 2g    h)  7-isopropyl-1,2,4-benzo-                            47     186-188        triazin-3-ol 1-oxide from  (isopropanol)        compound of Example 2h    i)  7-t-butyl-1,2,4-benzo-                            24     210-212        triazin-3-ol 1-oxide from  (ether)        compound of Example 2i    j)  6,7-dimethyl-1,2,4-benzo-                            52     230-232        triazin-3-ol 1-oxide from  (ethyl acetate)        compound of Example 2j    k)  5-methoxy-1,2,4-benzo-                            42     241-243        triazin-3-ol 1-oxide from  (ethyl acetate)        compound of Example 2k    l)  7-methoxy-1,2,4-benzo-                            84     242-244        triazin-3-ol 1-oxide from  (water)        compound of Example 2l    m)  7-(1,1-dimethylcyano-                            75     216-218        methyl)-1,2,4-benzo-       (water)        triazin-3-ol 1-oxide        from compound of Example 2m    n)  7-hydroxy-1,2,4-benzo                            57     264-265        triazin-3-ol 1-oxide from  (ether)        compound of Example 2n    ______________________________________

EXAMPLE 9 7-(1,1-Dimethylcyanomethyl)-1,2,4-benzotriazin-3-ol

A solution of 1.5 g (6.5 mmol) of compound of Example 8m in 30 mlglacial acetic acid is mixed at 85° C. within 30 min with 1.8 g ironpowder. One filters off with suction hot, after-washes with hot glacialacetic acid, evaporates the filtrate and chromatographs on silica gel.After elution with ethyl acetate/methanol 3:1 and trituration withether, there remain 0.8 g of title compound (57% of theory) of the m.p.183°-185° C.

EXAMPLE 10 Inhibition of the PLA₂ activity

a) Inhibition of the human recombinant type II PLA₂) (=synovial PLA₂)

As typical respresentative of A PLA₂, there was selected for the testingthe synovial PLA₂ (dissertation of Rainer Muller, 1993; University ofRegensburg).

As representative of the compounds I or Ia, respectively, there wasinvestigated in more detail the compound from Example 1. In thefollowing Table, there is given dosage-dependent the percentage in vitroinhibition of PLA₂.

    ______________________________________    compound from Example                     PLA.sub.2 inhibition  %!    ______________________________________    1         100 μg/ml                         99    1          10 μg/ml                         60    1          1 μg/ml                         43    ______________________________________

b) Inhibition of the collagen-induced arachidonic acid

(AA) liberation from human thrombocytes

As further indicator of a PLA₂ inhibition, there is valid an inhibitionof the AA liberation from thrombocytes. For this purpose, thethrombocytes are equilibrated with ³ H-AA. This added radioactivearachidonic acid incorporates into membrane phospholipids. Subsequently,the thrombocytary PLA₂ is activated via the collagen receptor and ³ H-AAthus again liberated from the membrane phospholipids into the mediumwhere it can be measured.

In table 1 is shown that the representative selected compound of example1 inhibits dosage-dependent the collagen-induced AA liberation by up to91%.

    ______________________________________                    inhibition of the AA    compound of Example                    liberation  %!    ______________________________________    1         100 μg/ml                        91    1          10 μg/ml                        29    ______________________________________

We claim:
 1. A method of inhibiting PLA₂ in a mammal in need of suchinhibition comprising administering to said mammal a PLA₂ inhibitingeffective amount of a compound of formula I ##STR5## wherein R₁ ishydroxyl,R₂ and R₃, which are the same or different, are selected fromthe group consisting of hydrogen, chloro, C₁ -C₆ -alkyl, C₁ -C₆ -alkoxy,cyano-C₁ -C₆ -alkyl, and hydroxyl, and n is the number 0 or 1, theirtautomers, and salts with non-toxic acids or bases.
 2. The method ofclaim 1, wherein R₂ in 7- position is selected from the group consistingof hydrogen, chloro, C₁ -C₆ -alkyl, C₁ -C₆ -alkoxy, cyano- C₁ -C₆ -alkyland a hydroxyl group.
 3. The method of claim 1, wherein R₃ is selectedfrom the group consisting of hydrogen and a C₁ -C₆ -alkyl group.
 4. Acompound of the formula Ia ##STR6## wherein R₂ is in 7- position and isselected from the group consisting of hydrogen, C₁ -C₆ -alkyl, C₁ -C₆ -alkoxy, cyano-C₁ -C₆ -alkyl, and hydroxyl;R₃ is a hydrogen or C₁ -C₆-alkyl, wherein when R₂ is hydrogen, R₃ is a C₁ -C₆ -alkyl; and n is thenumber 0 or
 1. 5. The compound of claim 4, wherein R₂ is CH₃, R₃ ishydrogen and n is the number 0 or
 1. 6. A pharmaceutical compositioncomprising a compound of claim 4 and a pharmaceutically acceptablecarrier or adjuvant.
 7. A pharmaceutical composition comprising acompound of claim 5 and a pharmaceutically acceptable carrier oradjuvant.
 8. A method for reducing inflammation by inhibiting PLA₂ in amammal in need of such inhibition comprising administering to saidmammal an inflammation reducing effective amount of a compound offormula I ##STR7## wherein R₁ is hydroxyl,R₂ and R₃, which are the sameor different, are selected from the group consisting of hydrogen,chloro, C₁ -C₆ -alkyl, C₁ -C₆ -alkoxy, cyano-C₁ -C₆ -alkyl, andhydroxyl, and n is the number 0 or 1, their tautomers, and salts withnon-toxic acids or bases.
 9. The method of claim 8, wherein R₂ in 7-position is selected from the group consisting of hydrogen, chloro, C₁-C₆ -alkyl, C₁ -C₆ -alkoxy, cyano- C₁ -C₆ -alkyl and a hydroxyl group.10. The method of claim 8, wherein R₃ is hydrogen or C₁ -C₆ -alkylgroup.